Role of the Pbrm1 subunit and the PBAF complex in Schwann cell development

Myelin sheath formation in the peripheral nervous system and the ensuing saltatory conduction rely on differentiated Schwann cells. We have previously shown that transition of Schwann cells from an immature into a differentiated state requires Brg1 that serves as the central energy generating subunit in two related SWI/SNF-type chromatin remodelers, the BAF and the PBAF complex. Here we used conditional deletion of Pbrm1 to selectively interfere with the PBAF complex in Schwann cells. Despite efficient loss of Pbrm1 early during lineage progression, we failed to detect any substantial alterations in the number, proliferation or survival of immature Schwann cells as well as in their rate and timing of terminal differentiation. As a consequence, postnatal myelin formation in peripheral nerves appeared normal. There were no inflammatory alterations in the nerve or other signs of a peripheral neuropathy. We conclude from our study that Pbrm1 and very likely the PBAF complex are dispensable for proper Schwann cell development and that Schwann cell defects previously observed upon Brg1 deletion are mostly attributable to altered or absent function of the BAF complex.

www.nature.com/scientificreports/ Considering that all essential and constitutive subunits of BAF and PBAF complexes occur in developing Schwann cells 1,13,14 it is currently not clear, to what extent the previously observed defects in Schwann cell development after Brg1 deletion are due to impaired function of the Brg1-containing BAF complex as opposed to the equally Brg1-containing PBAF complex.
To shed light onto the role of the PBAF complex, we here chose to delete the essential PBAF subunit Pbrm1 from Schwann cells and study their development. From the absence of major alterations in lineage progression, precursor cell survival and expansion as well as terminal differentiation and myelination, we conclude that Pbrm1 is of minor relevance in developing Schwann cells. In comparison to PBAF complexes, the Brg1-containing BAF complexes therefore appear to be the major SWI/SNF-type chromatin remodelers in Schwann cells.

Pbrm1 expression in Schwann cells.
To be able to study Pbrm1 on the protein level, we generated an antibody against amino acids 1288-1392 of mouse Pbrm1. By immunohistochemistry, Pbrm1 was detected along embryonic spinal nerves from E12.5 until E18.5 and in sciatic nerves from birth into adulthood in all cells that were positive for the pan-Schwann cell marker Sox10 (Fig. 1A). Using co-immunohistochemistry with stage-specific markers, we were further able to show that Pbrm1 occurs in Sox2-positive immature Schwann cells as well as in Oct6-positive pro-myelinating and Egr2-positive myelinating Schwann cells (Fig. 1B). Published RNA-Seq data 13 (Fig. 1C) and our own quantitative RT-PCR experiments (Fig. 1D) furthermore confirmed that Pbrm1 is expressed in Schwann cells cultivated under proliferating and differentiating conditions with levels decreasing approximately by one third during differentiation.
Within the peripheral nerve, Pbrm1 was not restricted to Schwann cells, but equally occurred in other cell types. This included fibronectin-positive fibroblasts, α-smooth muscle actin-positive perivascular smooth muscle cells, Desmin-positive pericytes, Pecam-positive endothelial cells, Iba1-positive macrophages and CD3-positive T lymphocytes (Fig. 1E).
Phenotypic consequences of Schwann cell-specific Pbrm1 deletion. To study the role of Pbrm1 in Schwann cell development, we combined a Pbrm1 floxed allele 15 in the homozygous state with a Dhh::Cre that has been previously shown to efficiently delete in the Schwann cell lineage starting at the late precursor cell stage 16 . The resulting mice are referred to as Pbrm1 cKO mice.
To confirm efficient deletion, we first convinced ourselves of the specificity of our anti-Pbrm1 antibodies. In western blots and immunocytochemical stainings, our antisera but not the preimmune sera specifically recognized the protein in Pbrm1-transfected HEK293 cells ( Fig. 2A,B). Using these antisera, we determined a 97% deletion rate in sciatic nerve Schwann cells at P0 (Fig. 2C). Wildtype Pbrm1 transcripts were also reduced to 46% in the sciatic nerve of Pbrm1 cKO mice at P7, in line with the fact that Schwann cells constitute a bit less than half of the nerve-associated cells (Figs. 2D, 3D). In contrast, transcript amounts for Arid2, Brd7 and Phf10 as other characteristic components of the PBAF complex were comparable in the sciatic nerve of control and Pbrm1 cKO mice at P7, P14 and P60 (Fig. 2E). Expression levels of the BAF complex subunits Arid1b, Brd9 and Dpf1 also remained unaltered arguing against a compensatory upregulation (Fig. 2F).
Pbrm1 cKO mice were born at the expected Mendelian ratio of 25% in breedings of Pbrm1 fl/fl with Pbrm +/fl Dhh::Cre mice and exhibited a postnatal growth that was indistinguishable from control littermates (Fig. 2G,H). Grip strength and the ability to hold onto an inverted cage lid were inconspicuous. There were no signs of hindlimb clasping. By overall appearance and opacity, sciatic nerves of Pbrm1 cKO mice and age-matched controls were indistinguishable as exemplified for P21 (Fig. 2I).

Characterization of Schwann cell development in the absence of Pbrm1. To characterize
Schwann cell development in more detail, we first determined sciatic nerve size and overall cell number from birth until two months of age. Judged by the area of the tibial branch in sciatic nerve sections at upper thigh levels and the number of DAPI-stained nuclei, no significant differences were evident between sciatic nerves of control and Pbrm1 cKO mice at any given time point (Fig. 3A,B).
Next we determined Schwann cell numbers during the postnatal period of active myelination in the sciatic nerve of Pbrm1 cKO mice. Using Sox10 as a marker for all Schwann cells, we counted similar absolute cell numbers from the day of birth until two months of age (Fig. 3C). By comparing Schwann cell to total cell numbers, Figure 1. Pbrm1 occurrence in peripheral nerves and Schwann cells. (A) Co-immunohistochemistry with antibodies directed against Pbrm1 (red, left and middle panels) and Sox10 (green, middle panels) in embryonic spinal nerves (boundaries depicted by dotted lines) at E12.5, E14.5, E16.5 and E18.5 as well as postnatal sciatic nerves at P0, P7, P14, P21 and P60. Nuclei were counterstained with DAPI (blue, right panels). (B) Co-immunohistochemistry with antibodies directed against Pbrm1 (red, left and right panels) and Sox2, Oct6 and Egr2 as stage-specific Schwann cell markers (green, middle and right panels) at E14.5 and E18.5 as indicated. (C, D) Analysis of Pbrm1 expression in Schwann cells cultured under proliferating (hatched bars) and differentiation (black bars) conditions according to published RNA-Seq data (GSE101153, presented as absolute number of counts for n = 3, in C) and quantitative RT-PCR (with average normalized expression levels in proliferating cells set to 1 and shown as mean ± SEM for n = 4, in D). Statistical significance was determined by unpaired two-tailed Student's t-test (**P ≤ 0.01). (E) Co-immunohistochemistry with antibodies directed against Pbrm1 (red) and various markers for other nerve-associated cell types (green), including fibronectin (FN), α-smooth muscle actin (Sma), Desmin, Pecam, Iba and CD3 in sciatic nerve tissue at P14 Scale bars: 10 µm (A, B, E). www.nature.com/scientificreports/ it became evident that the overall contribution of Schwann cells to the nerve cell population was comparable between Pbrm1 cKO mice and controls (Fig. 3D). Distribution of Schwann cells throughout the nerve of Pbrm1 cKO mice was furthermore indistinguishable from controls as judged from the Sox10 stainings (Fig. 3E). In line with similar absolute and relative Schwann cell numbers, we also failed to detect substantial changes in the number of proliferating Schwann cells regardless of whether Ki67 or Mcm2 was used as marker (Fig. 3F,G). Cell death rates in sciatic nerves of Pbrm1 cKO mice were not significantly different from controls as determined by TUNEL and staining for cleaved caspase 3-positive cells (Fig. 3H,I). We conclude from these results that there are no substantial differences in Schwann cell numbers or their proliferation and survival upon loss of Pbrm1.

Scientific Reports
To characterize Schwann cell lineage progression, we next studied the occurrence of stage-specific markers in sciatic nerves of Pbrm1 cKO mice during the first two postnatal months on transcript levels. By quantitative RT-PCR, transcript levels for the immature Schwann cell marker Sox2, the pro-myelinating Schwann cell marker     www.nature.com/scientificreports/ Oct6 and the myelinating Schwann cell marker Egr2 in sciatic nerves of Pbrm1 cko mice were comparable to controls at all time points analyzed during the first two months after birth (Fig. 4A). Immunohistochemical studies confirmed a normal lineage progression. We were unable to detect significant differences in Oct6 protein expression between sciatic nerves of Pbrm1 cKO mice and age-matched controls (Fig. 4B). For this marker of the pro-myelinating stage, numbers were already elevated at P0 in both genotypes, increased even further at P7 and then steadily declined, reflecting the transition of pro-myelinating into myelinating Schwann cells. The rise of myelinating Schwann cells is also visible in the gradual increase of Egr2-positive cells from 25 ± 4 cells at P0 to 146 ± 7 cells at P14 in controls (Fig. 4C,D). From their peak at P14 and P21, numbers of Egr2-positive cells per nerve section slightly decreased at older ages concomitant with the substantial longitudinal extension of the sciatic nerve. Again, no substantial differences were detectable in Pbrm1 cKO mice (e.g. 20 ± 7 cells at P0 to 141 ± 12 cells at P14). We therefore conclude that lineage progression and stage specific marker gene expression are unaltered in Pbrm1-deficient Schwann cells.
Characterization of myelin gene expression and myelination in the nerve of Pbrm1 cKO mice. Considering that Schwann cells develop normally and on schedule in Pbrm1 cKO mice, we next turned our attention to the myelination process. First, we analyzed myelin gene expression. By quantitative RT-PCR, overall transcript levels for Mbp and Mpz were comparable in sciatic nerves of control and Pbrm1 cKO mice (Fig. 5A). In addition to similar transcript levels, numbers of Mbp-and Mpz-expressing Schwann cells were highly alike in the sciatic nerve of both genotypes at all analyzed time points during the active phase of myelination as well as in two months-old young adults as determined by immunohistochemistry and in situ hybridization (Fig. 5B-E). We therefore conclude that myelin gene expression remains unaltered even after Pbrm1 loss in Schwann cells.
For visualization of myelin structures, PPD stainings were performed on sciatic nerves at P21 (Fig. 6A). Light microscopic inspection of these stainings did not point to any obvious alterations in the degree of myelination and the appearance of myelin in sciatic nerves of Pbrm1 cKO mice. Quantifications confirmed that the percentage of www.nature.com/scientificreports/ myelinated axons among axons bigger than 1 µm was close to 100% in both genotypes at P21 (Fig. 6B). Neither binning of g-ratios by axon diameter nor a scatter blot of single fiber g-ratios revealed substantial deviations in Pbrm1 cKO mice from controls (Fig. 6C,D). The mean g-ratio was determined as 0.66 in controls and 0.67 in Pbrm1 cKO mice (Fig. 6E). Size distribution of myelinated axons was likewise comparable between both genotypes (Fig. 6F). Electron microscopic analysis confirmed a normal ultrastructure of myelin sheaths around largecaliber axons as well as intact Remak bundles (Fig. 6G). There were no signs of activated macrophages. This was corroborated by normal numbers of Iba1-positive macrophages in immunohistochemical stainings at all times of analysis (Fig. 6H,I). Thus, peripheral nerve myelination is largely normal even if Schwann cells lack Pbrm1.

Discussion
In this study we have analyzed the consequences of Dhh::Cre-dependent Pbrm1 deletion in developing Schwann cells and have found no substantial alteration in the timing of lineage progression, survival and proliferation as well as in terminal differentiation and myelination. Similar results were also obtained at P0 and P7 when Sox10::Cre or Cnp1::Cre were employed instead of Dhh::Cre to delete Pbrm1 in Schwann cells. Pbrm1 has been shown to be an essential component of the vertebrate PBAF complex 9,12 . Normal development of Pbrm1-deficient Schwann cells therefore argues that the PBAF complex is not essentially required as chromatin remodeler during Schwann cell development and myelination. The absence of major phenotypic alterations in Schwann cell development after Pbrm1 loss is in contrast to previous observations on the consequences of Brg1 deletion in Schwann cell development by the same Cre transgene 7 . Brg1 functions as the ATP-hydrolyzing and energy generating subunit in both the BAF and the PBAF complex 8 . Two scenarios can therefore explain the different phenotypes in the two mouse mutants. For one, it can be envisaged that Schwann cell development is only affected in the joint absence of the Brg1-containing BAF and PBAF complexes and that separate inactivation of either chromatin remodeler alone remains without major consequences. This would require each complex to largely compensate for the other. Alternatively, most of the effects previously observed after Brg1 deletion are due to its activity in the BAF complex rather than its activity in the PBAF complex.
Brg1 is recruited to Schwann cell-specific enhancers of the Oct6 and Egr2 genes as well as to enhancers of several myelin genes via Baf60a and its direct physical interaction with Sox10 7,17 . Baf60a is a subunit of the BAF complex but does not occur in the PBAF complex. Selective enhancer recruitment of the BAF complex may therefore explain its unique role in Schwann cell development.
In the central nervous system, oligodendrocytes represent the functional substitute of Schwann cells. In agreement with their divergent ontogenetic origin, Brg1 functions differently in oligodendrocytes and Schwann cells 7,18,19 and it is by no means clear how oligodendroglial Brg1 functions partition between BAF and PBAF complexes. Future studies will have to clarify the role of Pbrm1 and the PBAF complex in oligodendrocytes and their development.

Methods
Generation and validation of Pbrm1 antibodies. Antibodies were raised in rabbit and guinea pig against a peptide that corresponded to amino acids 1288-1392 of mouse Pbrm1 by Pineda Antikörper-Service (Berlin, Germany). The peptide was produced in E. coli BL21 pLysS bacteria from a pET28a plasmid after IPTG induction and purified from whole bacterial extracts under denaturing conditions by an aminoterminally fused 6xHis-tag.
Antiserum from the final bleed was tested in western blot and immunochemical applications ( Fig. 2A,B). For western blots, whole cell extracts from pCMV5-Pbrm1 transfected HEK293 cells were size-fractionated on a standard 10% polyacrylamide-sodiumdodecylsulfate-gel, transferred to a nitrocellulose membrane and incubated with antiserum at a 1:3000 dilution as previously described 7 . Antibody-specific signal detection was by horseradish peroxidase coupled to protein A and luminol reagent. For immunocytochemical stainings, HEK293 cells were seeded on cover slips, transfected with pCMV5-Pbrm1-GFP or pCMV5-GFP, paraformaldehyde-fixed after 48 h and analyzed for GFP and Pbrm1 expression by stepwise incubation with anti-GFP and anti-Pbrm1 antisera followed by fluorophore-coupled secondary antibodies (see section on immunohistochemical analysis) according to previously published protocols 7 . Antiserum specificity was further confirmed by immunohistochemistry on control and Pbrm1-deleted tissues (Fig. 2C). Overview of sciatic nerve tissue from control (Ctrl) and Pbrm1 cKO mice at P21 after PPD staining. (B) Quantification of the percentage of myelinated axons in sciatic nerves of control and Pbrm1 cKO mice at P21 (shown as mean ± SEM, n = 3 per genotype). (C-E) G-ratio determination for myelinated axons in sciatic nerves of control and Pbrm1 cKO mice after binning by axon diameter (C), as scatter blot for single fibers (D, based on n = 150 fibers per genotype) or as average (E, shown as mean ± SEM, n = 3 per genotype). (F) Percental size distribution of axons (larger than 1 µm) in sciatic nerves of control and Pbrm1 cKO mice at P21 after binning by diameter (n = 150 fibers per genotype). (G) High resolution electron microscopic pictures of sciatic nerve tissue from control and Pbrm1 cKO mice at P21 depicting a representative Remak bundle (upper panels) and myelinated large calibre axons (lower panels). (H) Determination of Iba1-positive macrophages per sciatic nerve cross-section in control and Pbrm1 cKO mice from P0 until P60 (mean ± SEM, n = 3 per genotype). (I) Immunohistochemical stainings of nerve sections of control and Pbrm1 cKO mice from P0 until P60 with antibodies directed against Iba1. Scale bar: 10 µm (A, I), 3 µm (G). Statistical significance between genotypes was determined separately for each time point, bin and marker by unpaired two-tailed Student's t-test. However, no significant difference was detected. were obtained from the Jackson Laboratories (Bar Harbor, ME, USA) and were bred with mice expressing a Dhh::Cre transgene 16 . Genotyping was performed by PCR using 5′-GAC ATG GCT TCT CCC AAA CT-3′and  5′-TGC AAC TCT TTG TCC TTA CACG-3′ as primers in 33 cycles of 30 s 94 °C, 40 s 60 °C and 60 s 72 °C for  Pbrm1, and 5′-ATG CTG TTT CAC TGG TTA TG-3′ and 5′-ATT GCC CCT GTT TCA CTA TC-3′  Immunohistochemical analysis and in situ hybridization. Embryos and dissected sciatic nerve tissue underwent fixation in 4% paraformaldehyde, cryoprotection in 30% sucrose, embedding in Tissue Freezing Medium (Leica), and cryotome sectioning at 10 µm thickness before staining with the following primary antibodies: anti-Sox10 goat antiserum (1:3000 dilution, RRID:AB_2891326) 21   Histology and electron microscopy. Dissected sciatic nerves were successively incubated in cacodylatebuffered fixative containing 2.5% paraformaldehyde and 2.5% glutaraldehyde and in cacodylate-buffered 1% osmium ferrocyanide, dehydrated and embedded in Epon resin. Sectioning was at 1 µm for para-phenylenediamine (PPD) stainings (Carl Roth, Karlsruhe, Germany) or at 50 nm for staining with uranyl acetate and lead citrate. PPD stainings were analyzed by a Leica DMR microscope and used to determine the number of myelinated axons. Stainings with uranyl acetate and lead citrate were studied under a Zeiss Libra electron microscope (Carl Zeiss, Inc.) for analysis of nerve ultrastructure. Axonal perimeter as well as the perimeter of the surrounding myelin sheath were measured using Fiji 24 . Perimeters were subsequently used to calculate the respective diameters and determine the g-ratios (defined as the ratio of the inner axonal diameter and the outer, myelinated axonal diameter.